Microinjection of covalently cross-linked actin oligomers causes disruption of existing actin filament architecture in PtK2 cells.
Experiments were performed to determine the effects of interrupting the flux of actin monomers between unpolymerised and polymerised pools in PtK2 cells by (1) microinjecting exogenous polymerisation nuclei and (2) blocking endogenous assembly sites with low concentrations of cytochalasin D. Fluorescent actin oligomers were prepared by glutaraldehyde cross-linking F-actin derivatised at cysteine-374 with 5-iodoacetamido-fluorescein. These oligomers caused rapid nucleation of polymerisation of pyrene-labelled actin in vitro. Different numbers of polymerisation nuclei were injected into PtK2 cells and the cells were incubated for various times. Microinjection of between 1.2 X 10(4) and 1.8 X 10(4) nuclei per cell resulted in the complete disassembly of existing actin filament structures in nearly half of the cells within 15 min. Existing structures in such cells were replaced by foci of polymerised actin, which co-localised with concentrations of nuclei. Injection of increasing numbers of nuclei between 3 X 10(3) and 1.2 X 10(4) caused fragmentation of stress fibres in an increasing proportion of cells, whereas injection of less than 3 X 10(3) caused no obvious effects even over a 90 min incubation period. These data indicate that the degree of disruption of stress fibres was a function of the number of nuclei injected, but that it was less dependent on the incubation time. The minimum number of injected nuclei causing complete disruption of actin filament structures provides an estimate for the number of endogenous nuclei involved in filament turnover, whereas the minimum period for reorganisation (about 15min) implies a maximum time for the complete turnover of actin in the system.(ABSTRACT TRUNCATED AT 250 WORDS)
Handel
S E
SE
Department of Human Anatomy and Cell Biology, University of Liverpool, UK.
Hendry
K A
KA
Sheterline
P
P
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000502:physiology* / Q000648:ultrastructure; D000818:Animals; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D015638:Cytochalasin D / Q000494:pharmacology; D046911:Macromolecular Substances; D008845:Microinjections
D000199:Actins; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D015638:Cytochalasin D